i) Field of the Invention
This invention relates to monoclonal antibodies capable of specifically interacting with a human-derived thrombin-binding substance and also to their utilization.
ii) Description of the Related Art
The cell fusion technology has been developed rapidly since the report of Kohler and Milstein [Nature, 495-497 (1975)]. It has been known that a hybridoma obtained by fusing mammalian spleen cells and myeloma cells produces various antibodies depending on the characteristics of the spleen cells employed. It has also been attempted to form a hybridoma, which produces a monoclonal antibody against various biological substances such as proteins and hormones, by effecting cloning based on the characteristics of the hybridoma and also to produce the monoclonal antibody [E. Dale Servier et al., Clinical Chemistry, 27(11), 1779-1806 (1981)].
In the meantime, the present applicant previously succeeded in isolating from the human placenta a thrombin-binding substance (hereinafter called "TM"), which binds thrombin and specifically enhances activation of Protein C, and purifying same (Japanese Patent Application Laid-Open No. 199819/1985). TM has the following properties and is hence useful as a medicine.
(1) Molecular weight: PA0 (2) Isoelectric point: PA0 (3) Affinity: PA0 (4) Activities: PA0 (5) Stability: PA0 Derived from the placenta: PA0 Derived from the lung: PA0 Derived from plasma or urine: PA0 Derived from hemangioendothelial cells or pulmonary carcinoma cells:
88,000.+-.10,000 in reduced state; and PA1 71,000.+-.10,000 in non-reduced state. PA1 pH 4.2.+-.0.5. PA1 Strong affinity to thrombin. PA1 (a) Capable of binding thrombin and activating Protein C. PA1 (b) Capable of prolonging the clotting time. PA1 Stable in a pH range of 2-10. PA1 Stable against treatment by a denaturing agent (sodium dodecylsulfate or urea) or pepsin. PA1 J. Biol. Chem., 259, 12246-12251 (1984). PA1 Thrombosis Research, 37, 353-364 (1985). PA1 EP182929A. PA1 WO 87/0050. PA1 J. Clin. Invest., 76, 2178-2181 (1985). PA1 J. Biol. Chem., 260, 15432-15438 (1985).
Further, the human-derived TM has thereafter been reported in the following articles, patent publications, etc.
TM is however contained only in a trace amount in the placenta, and no sufficient specific binding is established with TM in various chromatographic techniques which are employed routinely. It is hence difficult to obtain TM in a highly pure form. Moreover, such conventional procedures require many steps and are unable to achieve any satisfactory recovery rate.
It has therefore been desired to develop a specific purification process for obtaining high-purity TM easily in a high yield.
It has also been desired to develop a high-sensitivity assay for TM as a means for the elucidation of the mechanism of the action of TM and also for the measurement of its blood level.
I. Maruyama, et al. have already prepared an anti-TM monoclonal antibody by using TM derived from the human placenta and investigated whether the antibody would inhibit the activation of Protein C caused as a result of binding of thrombin by TM derived from hemangioendothelial cells or pulmonary carcinoma cells [J. Biol. Chem., 260, 15432-15438 (1985)]. However, the discussion of this article only shows the property of the monoclonal antibody that it can recognize a thrombin-binding site of TM. It discloses nothing about other biological and physicochemical properties of the monoclonal antibody.